One means for improving a plant is a “transformation technique”, in which a desired recombinant gene for modifying a character is introduced into a plant. Efficient and quick transformation techniques are extremely important for the molecular breeding of useful plants, in particular grain crops, which are important staple foods.
A majority of grain crops (e.g., rice, wheat, barley, and corn) are classified as monocotyledons. Various transformation techniques for transforming monocotyledons have hitherto been developed. Transformation techniques are generally classified into direct transformation techniques and indirect transformation techniques.
Examples of direct transformation techniques include electroporation techniques (see Shimamoto K. et al., Nature, 338:274-276, 1989; and Rhodes C. A. et al., Science, 240:204-207, 1989), particle gun techniques (see Christou P. et al., Bio/Technology 9:957-962, 1991) and polyethylene glycol (PEG) technique (see Datta, S. K. et al., Bio/Technology, 8:736-740, 1990). Electroporation techniques and particle gun techniques have been generally used as methods for transforming monocotyledons which can achieve a relatively efficient gene introduction.
An example of an indirect transformation technique is an Agrobacterium-mediated transformation technique (hereinafter, this may also be referred to as an “Agrobacterium transformation technique”). Agrobacteria are a kind of plant pathogenic bacteria. Agrobacteria are characterized in that, when a plant is infected therewith, a T-DNA region which is present on the plasmids that Agrobacteria have (e.g., Ti plasmid or Ri plasmid) is incorporated into the plant. The Agrobacterium transformation technique utilizes the incorporation of the T-DNA region into plants as a means for introducing genes into plants. In short, a plant is infected with an Agrobacterium which contains a desired recombinant gene. After infection, a desired recombinant gene is transferred from the Agrobacterium into plant cells so as to be incorporated into the plant genome.
The Agrobacterium transformation technique is sufficiently established so far as dicotyledons are concerned. A large number of stable transformed plants have already been created which express desired recombinant genes.
On the contrary, it has conventionally been recognized as difficult to apply the Agrobacterium transformation technique to monocotyledons. For example, Portrykus et al. (BIO/TECHNOLOGY, 535-542, 1990) report that monocotyledons cannot be infected with Agrobacteria. On the other hand, a great deal of attempts have been made to transform monocotyledons by using Agrobacteria, which have shed light to the possibility of applying the Agrobacterium transformation technique to monocotyledons.
For example, Raineri et al. took the blastodisk of rice, scarred it, and placed it on a medium which induces dedifferentiation; a few days later, the blastodisk portion was infected with an Agrobacterium. As a result, although normally regenerated plant bodies were not obtained, calluses having a foreign gene introduced therein were successfully induced (see Raineri, D. M. et al., Bio/Technology, 8:33-38, 1990).
The pamphlet of International Publication No. WO94/00977 discloses an Agrobacterium transformation technique for rice and corn. According to this method, it is necessary to employ cultured tissue (e.g., calluses), which is in the process of dedifferentiation or which have completed dedifferentiation, as a plant sample to be transformed by an Agrobacterium. Therefore, prior to infection with an Agrobacterium, it takes 3 to 4 weeks to induce dedifferentiation in order to produce dedifferentiated culture tissue from a plant sample to be transformed (e.g., a leaf section).
The pamphlet of International Publication No. WO95/06722 discloses a method which infects an immature germ of rice or corn with an Agrobacterium. However, it is quite cumbersome to isolate immature germs.
Accordingly, it will contribute to the molecular breeding of useful monocotyledons, including grain crops such as rice, if a quicker and efficient Agrobacterium transformation technique for monocotyledons becomes available.